Tumor cell killing by in vitro affinity-matured recombinant human monoclonal antibodies

We have developed a method that allows the rapid improvement of the affinit y of phage-displayed antibody fragments by selection on intact eukaryotic c ells.;A single chain Fv fragment, specific for the tumor-associated Ep-Cam molecule, was mutagenized by shuffling of the immunoglobulin light chain va riable region and DNA shuffling of both heavy and light chain variable regi ons. Higher-affinity mutants were selected from small phage display librari es by cell panning under stringent conditions. When converted to an intact fully human antibody, the mutagenized anti-tumor monoclonal antibody displa yed an affinity of 0.4 nM, a 15-fold improvement over the affinity of the o riginal antibody. Compared to previously reported affinity maturation schem es, panning on intact cells does not require purified targets for selection and may be particularly useful when the target molecule can not be express ed as a recombinant molecule or easily purified without disrupting its nati ve configuration. In vitro tumor cell killing assays demonstrated an improv ed performance of the higher-affinity antibody in complement-mediated tumor cell killing. In contrast, the lower-affinity antibody performed somewhat better in antibody-dependent cellular cytotoxicity assays and penetrated be tter in multicell spheroids of tumor cells, an in vitro model for the tumor penetration capacity of antibodies.